Membrane self-organization in generation of yeast polarity and organelle identity Intracellular compartmentalization and organization is a central feature of eukaryotic cells: it permits local increases in enzyme and substrate concentrations, facilitating critical biochemical reactions while isolating other parts of the cell from potentially harmful processes. We are interested in determining how cells maintain stable, recognizable membrane substructures within the constantly shifting cellular environment. Most distinct organelles or membrane domains contain unique markers of identity such as specific phosphoinositides (PIs) or GTPase species. How do these structures arise and how are their molecular identities stably established? How does positive feedback contribute to establishment of this distinct identity? Our specific aims are to: 1) create synthetic membrane organelles or polarized structures in S. cerevisiae using a novel phosphoinositide (PI) species not normally found in yeast. Yeast lack the PI species PI(3,4,5)P3, found in higher eukaryotes. We will focus on spatially targeting the lipid kinases and phosphatases that produce or degrade this species in order to determine which pathways are necessary to either establish a polarized PI(3,4,5)P3 membrane domain or a PI(3,4,5)P3 tagged organelle. By introducing a new PI species in a controlled manner, we will separate the role of PIs in organelle identity from that of other cellular components and illuminate the minimal requirements in the process of organelle biogenesis and the evolution/diversification of the secretory pathway. 2) Quantitatively analyze the generation of natural yeast polarization using light-controlled recruitment of Cdc42 (a small GTPase) and its GEF, Cdc24. Endogenous yeast polarized structures are marked by the active form of the GTPase Cdc42, but the precise feedback architectures that lead to stable polarity establishment are poorly understood. Using a newly developed light-activated protein recruitment system (from the Lim and Voigt labs) we will precisely modulate the spatio-temporal recruitment of Cdc42 and Cdc24. This approach will allow us to control and observe the kinetics of polarization and to dissect the regulatory feedback pathways facilitating step-like switching between stable membrane organization states. PUBLIC HEALTH RELEVANCE: Membrane self-organization plays a crucial role in the maintenance of normal cellular processes and sequestration of biochemical reactions. Disruption of stable membrane domains, such as a polarity defect in epithelial cells, leads to pathology such as polycystic kidney disease. Our goal is determination and characterization of the guiding principles of membrane self-organization, including the relative roles played by spatial and temporal recruitment and positive feedback.